Threshold glucose detection in urine

ABSTRACT

Methods are provided for glucose detection and quantification in urine. The methods include selecting a test device comprising a matrix impregnated with a chromogenic indicator mixture, locating the test device to promote incidental contact of same with animal urine, reading a developed indicator color after the device has been wetted with urine, and determining the animal&#39;s urine glucose concentration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/232,123, filed Aug. 30, 2002 now U.S. Pat. No. 6,599,474 which is adivisional of U.S. application Ser. No. 09/883,874, filed Jun. 18, 2001,now U.S. Pat. No. 6,444,169, herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a dry chemistry test device for glucosedetection in liquid samples. More particularly, the invention isdirected to a test device for threshold detection and quantification ofglucose in urine and to methods of making and using the same.

BACKGROUND AND SUMMARY OF THE INVENTION

The detection of glucose levels is important in the treatment ofdiabetes. Test devices for glucose testing of the blood and urine ofhuman diabetics, including many dry chemistry test devices or teststrips, are described in numerous papers and patents. Glucose tests havebeen available in a dry test strip format for more than three decades.Analysis of blood and urine using test strips plays an important role inthe diagnosis and treatment of human diabetes.

The history of dry chemistry disposable test-devices for glucosedetection is summarized in U.S. Pat. Nos. 3,964,871 and 5,563,042. Quickand simple glucose determination in body fluids using dry chemistry testdevices under field conditions is important for the early detection,diagnosis, and control of both human and animal diabetes. Keyrequirements for dry chemistry colorimetric tests are to detect glucosein a wide range of concentrations and to achieve easily discernablecolor distinctions at variously defined glucose levels.

For detection of relatively low glucose levels with test strips, severalreagents have been proposed. U.S. Pat. No. 3,886,045 describes anon-toxic indicator comprising 4-aminoantipyrene and phenolic compoundsthat can form colored quinones in the presence of glucose. In U.S. Pat.No. 4,427,770, there is described a similar formulation using4-aminoantipyrene, in a two pad test format to detect a wide range ofglucose levels. U.S. Pat. No. 5,824,491 reports that 4-aminoantipyreneproduces a gradual concentration/reflectance response from 0-400 mg/dL,with a linear concentration/reflectance response in the 150-400 mg/dLglucose range.

A sensitive and stable glucose test device was also described in U.S.Pat. No. 5,183,742. The test-device comprises a support having printedor coated detection reagent region on a surface. However, with only oneperoxidatively active indicator, it proved difficult to obtain welldiscernable color changes over the broad range of 20-500 mg/dL.

Other indicator types have been used for detection and quantification ofrelatively high glucose levels and of a relatively wide range of glucoselevels. In U.S. Pat. Nos. 4,303,753 and 4,340,669, an indicator incombination with a polymer (iodide +poly(vinylpyrrolidone)) is describedfor high range (500-1000 and 1000-10,000 mg/dL) glucose detection.Iodide salts have also been used in conjunction with a blue dye thatfades in the presence of hydrogen peroxide. See, e.g., U.S. Pat. No.3,814,668. The two components act in concert to provide a continuum ofindicator colors.

A threshold color control system was described in U.S. Pat. No.5,036,000. The system was designed for measurement of NAD(P)H. Analytesreact to form NAD(P)H which is, in turn, oxidized by a chromogen.Non-chromogen competing compounds, such as Fe(III) compounds, are addedto prevent a visible color change until a predetermined amount ofNAD(P)H is exceeded. When the predetermined amount of NAD(P)H isexceeded, a single, easy to discern, color change is produced.

The above-mentioned reagents and test devices were generally designedfor human urine testing. However, diabetes also afflicts animals. Forexample, feline diabetes is quite common, affecting about 0.2% to 1.0%of the cat population. In this affliction, glucose levels in the bloodmust be controlled. As with humans afflicted with diabetes,hyperglycemic cats are treated with insulin injections. While insulintreats the hyperglycemia, proper treatment of diabetes also requiresperiodic monitoring of glucose levels. Since elevated glucose levels inblood usually lead to elevated glucose levels in urine, periodicmonitoring of glucose levels in the urine would provide needed screeningfor proper animal treatment.

While both the patent and non-patent literature is replete withreference to glucose testing systems and devices, few dry chemistryglucose tests have been developed specifically for testing the bodyfluids of animals. European Pat. No. 0 060 133 describes a method anddevice for nonenzymatic glucose measurement in animal body fluids.Recently, a test strip product (Petstix™ 7, Bayer Agricultural Division(Etobicoke, Ontario, Canada)) for animal urine testing (includingglucose test) was introduced in the market. Petstix 7 uses the samecolor blocks as N-Multistix™ SG (Bayer Diagnostics Division (Elkhart,Ind.)), which is marked with U.S. Pat. No. 3,814,668.

There are several limitations and specific requirements for animal urinetesting with test strips. For example, there are salient compositionaldifferences between cat urine and human urine. One such difference isthat cat urine has a significantly higher specific gravity than doeshuman urine. Because of this difference, false readings may be obtainedif test strips designed for human use are used for glucose detection incats. Another limitation involves the problems in collecting a urinesample from the animal. In use with animal body fluids, it is alsopreferable to have a test device wherein the results are not dependentupon the amount of sample applied and the time of exposure to ambientconditions prior to activation, and wherein the results are stable for aprolonged period of time subsequent to activation.

SUMMARY OF THE INVENTION

The present invention is directed to a method, reagent composition, anddry chemistry test device for threshold glucose detection, particularlyin cats. The device is in the form of an indicator impregnated substratethat is preferably cut into pieces suitable for being distributed ontocat litter. The indicator/substrate combination is selected so thatafter being wetted by animal urine, the indicator result remainsdiscernable for a sufficient period of time to allow it to be observedby the pet owner or caretaker. Thus, the preferred indicator compositionshould not only be able to withstand the ambient conditions of a litterbox and prolonged exposure to these conditions until activation byanimal urine, but it should also have good stability even afteractivation with urine. The indicator reagent preferably should also beselected to indicate whether the reagent device has been contacted byanimal urine, regardless of whether abnormal glucose levels are present.

The reagent composition used in the present invention is based on astandard glucose enzymatic system including glucose oxidase andperoxidase, and a combination of peroxidatively active indicators(chromogens). The combination of chromogens of the present inventionprovides for good color differentiation corresponding to a variety ofurine glucose concentrations. Furthermore, glucose detection in caturine is preferably based on an established sensitivity threshold orconcentration threshold. At glucose concentrations less than thethreshold, there would be no discernible color change on the testdevice. To achieve the desired sensitivity threshold, in addition to thecombination of chromogens, the reagent composition includes a scavengerof peroxide/oxidized chromogens. The reagent composition undergoes acolor change only when exposed to urine having a glucose concentrationat or above the threshold level, and, when such occurs, easily visuallydiscernible color changes indicate higher glucose concentrations.

In one preferred embodiment the test device is prepared using acellulose paper substrate impregnated with a solution of the reagentcomposition. The paper is then dried and cut into small but visuallydetectable pieces to provide a device that may be distributed on catlitter. The inactivated device remains glucose sensitive for severaldays even at high humidity. After contact with cat urine, a colorindicative of glucose concentration develops and persists for a periodof time sufficient to allow the pet caretaker to observe the colordevelopment. Color stability is achieved by incorporating stabilizersand selected chromogens into the reagent composition.

Thus, one aspect of the invention includes a dry chemistry device forglucose detection and quantification in urine, said device comprising achromogenic indicator mixture comprising a first indicator capable ofcolor development to indicate the presence of a low-to-mediumconcentration of glucose, and a second indicator capable of colordevelopment to indicate the presence of a higher concentration ofglucose, wherein the first indicator also prevents color development ofthe second indicator unless the higher concentration of glucose ispresent in the urine, and a carrier impregnated with the chromogenicindicator mixture.

In a preferred embodiment of this invention, a scavenger is provided toprevent color development when the glucose concentration is below athreshold concentration. Therefore, another aspect of the invention is adevice for detection and quantification of glucose in urine comprising achromogenic indicator mixture comprising a first chromogenic indicatorreagent combination capable of color development to indicate thepresence of a lower concentration of glucose and a second chromogenicindicator reagent combination capable of color development to indicatethe presence of a higher concentration of glucose, wherein the firstchromogenic indicator prevents color development of the secondchromogenic indicator unless the higher concentration of glucose ispresent, a scavenger that prevents color development of the firstindicator reagent composition unless a threshold concentration ofglucose is present, and a matrix impregnated with the chromogenicindicator mixture.

A third aspect of this invention is a method for determining urineglucose concentration in an animal comprising the steps of selecting atest device comprising a matrix impregnated with a chromogenic indicatormixture comprising a first chromogenic indicator reagent combinationcapable of color development to indicate the presence of a lowerconcentration of glucose and a second indicator reagent combinationcapable of color development to indicate the presence of a higherconcentration of glucose, wherein the first chromogenic indicatorreagent combination prevents color development of the second indicatorunless the higher concentration of glucose is present, locating the testdevice to promote incidental contact of same with animal urine, readinga developed indicator color after the device has been wetted with urine,and comparing the developed color to a standard color chart anddetermining the animal's urine glucose concentration.

Another aspect of this invention is a test kit for determining theconcentration of glucose present in a sample of animal urine, the testkit comprising a substrate impregnated with a first chromogenicindicator reagent combination capable of color development to indicatethe presence of a lower concentration of glucose, and a secondchromogenic indicator reagent combination capable of color developmentto indicate the presence of a higher concentration of glucose, whereinthe first chromogenic indicator prevents color development of the secondchromogenic indicator unless the higher concentration of glucose ispresent in the urine sample; and color chart indicating urine glucoseconcentrations corresponding to a plurality of discernible colors.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of preferred embodiments exemplifying the best mode ofcarrying out the invention as presently perceived.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method, reagent composition, anddry chemistry test device for glucose detection. While the invention issuitable for urine glucose detection generally, it is particularlysuitable for urine glucose detection in cats. Preferably, the device isused for detection of glucose concentrations above a specific thresholdlevel. In one preferred embodiment, the test device is formed asconfetti-like material for use as a litter box additive. The problemspresented by urine sample collection from cats and the relatively highspecific gravity of cat urine present unique performance demands for thepresent test device relative to similar test strips used for testing ofhuman diabetes.

The test device comprises an indicator mixture composition and a supportmatrix for the indicator composition. In a preferred embodiment, thesupport matrix comprises a piece of filter paper impregnated with theindicator composition, wherein the filter paper is of sufficientporosity and capillary affinity to allow an urinary sample from theanimal to migrate into the matrix and interact with the indicator. Thematrix may be a woven or a non-woven material and may include, but isnot limited to, cellulosic natural fiber materials of the type normallyused to make filter papers. Alternatively, organic polymer materials maybe used. Other natural or synthetic fiber matrix materials, either wovenor non-woven, may also be used.

It is preferable that the reagent indicator composition of the testdevice develop a threshold color response, wherein no discernible colorchange is produced unless the amount of glucose in the urine sampleexceeds a predetermined concentration. Such would enable an animal owneror caretaker to observe the presence of a color change, indicating apossible hyperglycemic problem. Only if the threshold concentration wereexceeded would the alerted caretaker need to investigate further todetermine the severity of the hyperglycemic condition.

It is also preferable for the color differentiation to be strong enoughto allow a reliable color detection and interpretation directly on thecat litter surface. Furthermore, the test device should remain glucosesensitive for at least several days, even at high humidity, after beingapplied on the cat litter surface. Additionally, the color signal of theurine activated device should be stable for at least several hours afterexposure to cat urine. Other preferred features of the present testdevices include that the color from the activated indicator reagentshould not leach onto cat litter under conditions of normal use andobserved test color should not depend on the amount of cat urineapplied.

In one embodiment, the matrix impregnated with the indicator compositionmay be cut into small confetti-like pieces, to be sprinkled on or mixedinto litter. The small pieces may be of various geometric shapes andtypically 0.01 to 1.0 inches in its longest dimension, more typically,0.1 to 0.5 inches in its longest dimension, and most typically squaresor diamonds 0.2 inches on a side. However, it should be understood thatthe present invention is not limited for use with cats, and other shapesand sizes may be appropriate for human use or for use with otheranimals. For instance, much larger test sheets (1.0 to 20 inches in thelongest dimension) may be suitable for use with paper-trained dogs. Thelarger sheets also may be placed under litter material in a cat litterbox or under similar material for use with other animals. The testdevice may be sealed, for example in a foil package, to prolong productshelf life for distribution, sale, and use.

The reagents of the present invention are based on formulations havingglucose oxidase, peroxidase, and chromogenic redox indicators as keycomponents. Glucose is oxidized to gluconic acid by atmospheric oxygenin a specific glucose oxidase-catalyzed reaction that produces hydrogenperoxide as a byproduct proportional to the amount of glucose:

In various reactions catalyzed by peroxidase, hydrogen peroxide reactswith the colorless form of the indicator reagent composition to producewater and an oxidized, colored form of the indicator. The oxidized formof the indicator may itself be a colored entity or more typically reactswith another component to provide a colored product indicative of someconcentration range of glucose in the tested sample. In the indicatormixture of the present invention two different indicator reagents areused; one to detect low/medium glucose levels (Ind 1) via Reaction 1,and another to provide a different colored signal at high glucose levels(Ind 2) via Reaction 2.

Reaction 1 uses an indicator reagent composition (Ind 1) that has arelatively rapid susceptibility to oxidation by peroxidase. Thus,Reaction 1 has a high reaction rate as compared to Reaction 2. In thepresence of low/medium glucose levels (preferably 150-300 mg/dL), acomponent of Ind 1 is oxidized and corresponding colors (for examplerose or red when Ind 1 is AAP/HBS) are developed. The device detectslow/medium glucose levels mainly on the basis of Reaction 1:

Suitable compositions for Ind 1 include 4-aminoantipyrene (AAP), or asalt thereof, and phenolic compounds, such as2-hydroxy-3,5-dichlorobenzenesulfonate (HBS), 3-methylcatechol,4-hydroxybenzenesulfonic acid, and 2,6-dimethylphenol. Other phenolicand aniline compounds capable of forming a colored compound with AAP areknown in the art and are within the scope of this invention. See, forexample, U.S. Pat. Nos. 3,886,045 and 4,427,770, hereby incorporated byreference. Other chromogenic aromatic compounds may be substituted forAAP. For example, 3-methylbenzothiazolinone hydrazone hydrochloride(MBTH) may be used. The combination of 1,3-phenylenediamine and MBTHproduces an intense dull red color when activated with hydrogenperoxide.

In one embodiment of this invention, Ind 2 comprises an iodide salt.While potassium iodide is preferred, other iodide salts may be used,including but not limited to sodium iodide and ammonium iodide. Theiodine that is released in Reaction 2 forms a colored complex withstarch and other substances, as known in the art. See, for example, U.S.Pat. No. 3,886,045, hereby incorporated by reference. Substances capableof forming a colored complex with iodine include starch, starchcomponents such as amylose or amylopectin, polyethylene glycol,polyvinylpyrrolidone, and polyvinyl alcohol. However, depending on theuse, care must be taken in selecting a suitable substance, as someotherwise appropriate substances may be toxic. Because starch isrelatively inexpensive, non-toxic, and provides good color, starch ispreferred. Thus, Reaction 2 using iodide/starch is as follows:

In the absence of any other indicator, iodide/starch produces acharacteristic blue color in the 50-300 mg/dL range, with increasingblue color with increased glucose concentration.

Reaction 2 has been observed to proceed more slowly than Reaction 1. Ina preferred embodiment, the total stoichiometric amount of Ind 1 in thereagent composition is equal to, or slightly exceeds, a low/mediumglucose level. In the presence of low/medium glucose levels, Ind 1quickly consumes the available peroxide and Reaction 2 does not proceed.When AAP/phenol is used, the only color change observed at lowerconcentrations is due to AAP/phenol. A similar scavenging effect is seenwith MBTH/1,3-phenylenediamine. At high glucose levels (preferably above300 mg/dL) all of Ind 1 is consumed and Ind 2 interacts withH₂O₂/peroxidase. As a result, a color specific for Ind 2 (for exampledark brown-black or dark blue when Ind 2 is starch/iodate) is developed.Thus, Ind 2 is used mainly to detect high glucose levels.

In a preferred embodiment, in addition to the double indicatorcomposition described above, a scavenger is used to provide a thresholddetection level for the device, wherein no color change is observed atglucose urine levels below the threshold concentration. In thispreferred embodiment, a scavenger is used to prevent a color change atvery low levels of glucose, as shown in Reaction 3:

In the presence of very low glucose levels (preferably less than 50mg/dL) the colored (oxidized) form of Ind 1 may be produced. However, ascavenger may be selected so that under conditions of low glucoseconcentration, the scavenger reacts with oxidized Ind 1 and transformsit back to the colorless reduced form. Thus, in these conditions nocolor is developed and only the background coloration can be seen.Alternatively, a scavenger may be used that out competes the Ind 1indicator for peroxide, preventing Reaction 1 from proceeding in lowlevels of glucose. In either case, predetermined amounts of thescavenger in the formulation correspond to a very low glucose level. Inthe presence of glucose levels greater than the capacity of thescavenger (preferably 50 mg/dL and more), the concentration of theoxidized Ind 1 exceeds the concentration of the scavenger, Ind 1 remainsin its oxidized form, and glucose is detected. Depending on theapplication, suitable scavengers include stannous chloride,thiosulfates, and mercaptans. Because it is non toxic, a preferredscavenger is cysteine.

Urine from diabetic cats undergoing insulin therapy should have someresidual urine glucose. Therefore, it is preferred that the test deviceindicate a level of 50 mg/dL, in order to detect or prevent an overdose.This threshold level may be varied for different animal or humanapplications.

The above-described combination of indicators and scavengers provides athreshold glucose detection, as well as various indicator colors in thelow-high urine glucose concentration range associated with diabetes incats. It should be stressed that the threshold color change may beshifted as needed for individual applications, particularly in the50-1000 mg/dL glucose range. Appropriate amounts of Ind 1 and scavengermay be chosen to produce a threshold color and an Ind 1/Ind 2 colorchange at desirable glucose levels.

It is preferred that the test device have good stability and be able towithstand ambient conditions of a litter box and prolonged exposure ofthese conditions until its activation by being wetted with animal urine.Also, it is preferred that the resultant color produced upon activationby the animal urine remain unchanged for a significant period of time.In this regard, the scavenger (for example, cysteine) included in thereagent formulation plays a dual role. The scavenger not onlyfacilitates color distinction between very low and low glucose levels,but it also provides an antioxidant function to protect the indicatorsfrom spontaneous oxidation during reagent preparation, storage, andapplication.

In a preferred embodiment of the invention, the reagent composition isformulated to contain a polysaccharide, such as Dextran, DEAE-Dextran,or Alginate, to improve enzyme stability and to maintain adequate testperformance of the test device during prolonged exposure to ambientconditions on cat litter. Furthermore, a film-forming polymer, such aspoly(vinyl alcohol), may be used in the reagent composition to aid inpreventing the indicator from leaching from the reagent when activatedwith cat urine.

The following examples are set forth to illustrate the principles andpractices of the present invention to one skilled in the art. They arenot intended to be restrictive but merely to be illustrative of theinvention.

EXAMPLE 1

TABLE 1 Reagent Formulation No 1 Components Amount, g/L Distilled WaterSubmix 1 for First Dip Starch 6.0 Submix 2 for Second Dip Glucoseoxidase 43 ku/L Peroxidase 36 ku/L Potassium Iodide 10.24-Aminoantipyrene 1.86 2-Hydroxy-3,5-dichlorobenzenesulfonate 0.8Na-salt Tartrazine (Acid Yellow) 0.05 L-Cysteine 0.2 Citric Acid 2.3Citrate, Na-salt 11.2 Poly(vinyl alcohol) 20.0 Dextran 10.0

Cellulose paper was impregnated with Submix 1 and dried. The resultingpaper was impregnated with Submix 2 and dried again. The test paperimpregnated with the entire set of the components was cut into smallconfetti-like pieces to obtain a final test device.

The test device pieces were distributed on the surface of cat litter andsamples of 60 microliters of cat urine having different glucoseconcentrations were applied to the pieces. The color developed wasanalyzed visually and instrumentally by reflectance spectrophotometry.

TABLE 2 Visual and instrumental color evaluations. Color DifferenceGlucose Levels Test Piece vs. Non-Activated mg/dL Colors Test Piece Notactivated Yellow — (not wetted by cat urine) Activated with 50 mg/dL orless Light Tan 9.37 (50 mg/dL) 150 Rose 19.78 300 Red 42.13 600 Darkbrown-black 56.30

Urine with a specific gravity of 1.030 was used and observed one hourafter activation. The color differences are given in CIELAB delta EUnits and are based on instrumental reflectance measurement. The colordifference units represent the relative distinction by a normal observerof the color at the indicated concentration compared to the yellow (notactivated) color.

EXAMPLE 2

TABLE 3 Reagent Formulation No. 2 Components Amount, g/L or activityunits Glucose oxidase 104.3 kilounits Peroxidase  56.9 kilounitsPotassium Iodide 9.92 Starch 6.40 4-Aminoantipyrene 2.423-Methylcatechol 1.48 L-Cysteine 1.7 Citric Acid 38.4 Imidazole 1.0Alginic Acid 5.0 Triton X-1000 2.5

Filter paper was dipped into this solution and dried. The paper was thencut into pieces. For Reagent Formulation No. 2, the color chart is asfollows:

Glucose Conc. (mg/dL) Test Piece Color 50 light yellow 150 very lightolive 300 olive drab 600 brown

The resultant test device was tested with cat urine containing glucoseconcentration levels of 50, 150, 300 and 600 mg/dL. Prior to applicationon the test device, the specific gravity of each of the cat urinesamples was adjusted with water to either 1.020 of 1.050. With the lowerspecific gravity urine samples, colors developed within a few minutes,with the color corresponding to the glucose level in the sample. Therate of color development with the higher specific gravity urine sampleswas slower than that of the lower specific gravity samples. However,after ten minutes, the colors of the higher specific gravity seriesappeared similar to the lower specific gravity series. Thus, with higherspecific gravity the rate of color development was slowed, but theresultant color was sufficiently distinct to identify the variousglucose levels.

EXAMPLE 3 Stability of the Reagent After Activation with Cat Urine

Using Reagent Formulation No. 1 according to Example 1, test readingswere done at various times after activation with cat urine (specificgravity 1.030). The resulting color was read visually and the glucoseconcentration was determined by color comparison with a predeterminedcolor chart. As shown in Table 4, the test readings are stable for atleast 8 hours after activation with cat urine.

TABLE 4 Hours after reagent Actual glucose urine level, mg/dL activationwith cat 0 150 300 600 urine Measured glucose urine level, mg/dL 1 0 155320 600 4 0 157 310 600 8 0 150 310 600

Effect of Cat Urine Specific Gravity

Using Reagent Formulation No. 1 according to Example 1, test readingswere done four hours after activation with cat urine having varyingspecific gravities. The resulting color was read visually and theglucose amount was evaluated by color comparison with a predeterminedcolor chart. As shown in Table 5, specific gravity has only minimaleffect on the results.

TABLE 5 Actual glucose urine level, mg/dL 150 300 600 Specific GravityMeasured glucose urine level, mg/dL 1.020 160 310 600 1.040 135 290 5851.050 127 275 555

Using Reagent Formulation No. 1 according to Example 1, test readingswere done one hour after activation with varying amounts of cat urine(specific gravity of 1.030). The resulting color was read visually andthe glucose concentration was determined by color comparison with apredetermined color chart. As shown in Table 6, the amount of urineapplied in the range of 60-150 microliters does not significantly affecttest readings. Also, it was noted that larger volumes of urine do notseem to cause significant leaching of indicators from test pieces.

TABLE 6 Microliters applied Actual glucose urine level, mg/dL to thetest-piece of 1 150 300 600 cm² Measured glucose urine level, mg/dL 30112 250 525 60 150 300 600 90 150 300 600 120 150 320 600 150 150 320600

Although the invention has been described in detail with reference topreferred embodiments, variations and modifications exist within thescope and spirit of the invention as described and defined in thefollowing claims.

What is claimed is:
 1. A method for determining urine glucoseconcentration in an animal comprising the steps of selecting a testdevice comprising a matrix impregnated with a chromogenic indicatormixture comprising a first chromogenic indicator reagent combination forcolor development to indicate the presence of a lower concentration ofglucose and a second indicator reagent combination for color developmentto indicate the presence of a higher concentration of glucose, whereinthe first chromogenic indicator reagent combination prevents colordevelopment of the second indicator unless the higher concentration ofglucose is present, locating the test device to promote incidentalcontact of same with animal urine, reading a developed indicator colorafter the device has been wetted with urine, and determining theanimal's urine glucose concentration.
 2. The method of claim 1 whereinthe animal is a cat, the test device is provided in confetti-likepieces, and the locating step includes distributing the confetti-likepieces on the surface of cat litter.
 3. The method of claim 2 whereinthe test device remains on the cat litter for several hours prior toactivation.
 4. The method of claim 2 wherein the comparing step occursseveral days after activation.
 5. The method of claim 1 wherein the testdevice is provided in a large sheet, and the locating step includesplacing the sheet on the floor.
 6. The method of claim 1 wherein thetest device is provided in a large sheet, and the locating step includesplacing the sheet under litter material.
 7. The method of claim 1wherein the determining step comprises determining urine glucoseconcentration over a range of 50-1000 mg/dL glucose.
 8. The method ofclaim 1 wherein the determining step comprises comparing the developedcolor to a standard color chart.
 9. The method of claim 1 wherein thethreshold level of glucose is about 50 mg/dL glucose.